Multidrug resistance (MDR) refers to a resistance to one type of drug as well as to other antitumor drugs with different structures and different targets. Its development may exist before chemotherapy (i.e. intrinsic resistance), or may develop during chemotherapy (i.e. acquired resistance). So far, the understanding of the mechanism of drug resistance is still very limited, increasing the outflow rate of anticancer drugs from tumor cells through the ATP-binding cassette (ABC) transport superfamily on the cell membrane is one of the most important mechanism. The ABC family is a group of transmembrane protein with ATP binding regions for transporting substrate in one direction, which performs membrane transport of multiple molecules by active transport. Three of the family members ABCB1/p-gp (p-glycoprotein, P-gp), ABCC1/MRP1 (Multidrug Resistance-associated Protein 1, MRP) and ABCG2/BCRP (Breast Cancer Resistance Protein, BCRP) are highly expressed in tumors with drug resistance (FIG. 1-1). By reducing the concentration of antitumor drugs in the cells, or redistributing antitumor drugs within the cells, these transmembrane proteins cause the development of drug resistance of tumor cells.
ABCG2/BCRP/MXR ABCG2 belongs to the G subfamily of the ABC transport protein family, has 655 amino acid residues and a molecular weight of 72 kD. It is localized on the cell membrane, has an ABC family characteristic hydrophilic motif at 1-400 residues, and has unique structural features: (1) consisting of one ATP-binding domain and hydrophobic carboxy-terminal transmembrane region with six alpha-helical transmembrane structures, which belongs to the half transport protein (McDevitt C A, Collins R F, Conway M, et al. Purification and 3D structural analysis of oligomeric human multidrug transporter ABCG2. Structure, 2006, 14(11):1623-1632); (2) localized on the cell membrane although belong to the half transporter; (3) the ATP-binding domain of ABCG2 is localized at the N-terminal while the transmembrane domain is localized at the C-terminal, and is structurally distinct from other subfamily transporters such as P-gp and MRP1 (FIG. 1-2); (4) a functional ABCG2 transporter requires two or more nucleotide binding domains and transmembrane domains to form a substrate transfer channel (Cheffer G L, Maliepaard M, Pijnenborg A C, et al. Breast cancer resistance protein is localized at the plasma membrane in mitoxantrone- and topotecan-resistant cell lines. Cancer Res, 2000, 60(10):2589-2593). Therefore, ABCG2, as a semi-transporter, is likely to function as a homodimer or oligomer. Due to the specific structure of ABCG2, it is determined that the drug resistance mediated by ABCG2 is different from those of p-gp and MRP1.
Tumor drug resistance mediated by ABCG2 belongs to the intrinsic resistance. Researchers have isolated the cell components expressing ABCG2 from stem cells and tumor cell lines. The isolated cells have a stem cell-like phenotype, and resistant to the antitumor drugs mitoxantrone, doxorubicin, rubidomycin and topotecan (Kenneth K W and Fu L W. Multidrug resistance transporters-roles in maintaining cancer stem-like cells. Stem Cells in Clinic and Research. Chapter 30, 720-746, edited by Ali Gholamrezanezhad, ISBN 978-953-307-797-0, Published: Aug. 23, 2011 under CC BY-NC-SA 3.0license; Tan B, Piwnica-Worms D, and Ratner L, Multidrug resistance transporters and modulation. Curr Opin Oncol, 2000; 12,450-8.). ABCG2 is highly expressed in undifferentiated human embryonic stem cells (ESC) and cancer stem cells (CSC) (Kenneth K W and Fu L W. Multidrug resistance transporters-roles in maintaining cancer stem-like cells. Stem Cells in Clinic and Research. Chapter 30, 720-746, edited by Ali Gholamrezanezhad, ISBN978-953-307-797-0, Published: Aug. 23, 2011 under CC BY-NC-SA 3.0license; Apati A, Orban T I, Varga N, Nemeth A, Schamberger A, Krizsik V, Erdelyi-Belle B, Homolya L, Varady G, Padanyi R, Karaszi E, Kemna E, Nemet K, Sarkadi B. High level functional expression of the ABCG2 multidrug transporter in undifferentiated human embryonic stem cells. Biochim Biophy Acta, 2008, 1778(12), 2700-2709), indicating that it has protective effects on ESC and CSC. Therefore, the CSC intrinsic drug resistance mediated by ABCG2 can be used as a target for CSC.
ABCG2 has important physiological functions in human body, which is characterized by blocking the permeability of drugs and toxins via expressing on the capillary endothelium of the blood-brain barrier and the placental barrier; the expression on the polar surface of small intestinal mucosal cells and colonic epithelial cells is associated with restricted absorption; the expression in liver and kidney tissues is associated with the elimination of drugs and toxins. ABCG2 acts as a multidrug resistance regulatory protein in breast cancer, colon cancer, small cell lung cancer, ovarian cancer, gastric and intestinal cancer, and malignant melanoma (Kathawala R J, Gupta P, Ashby C R Jr, Chen Z S. The modulation of ABC transporter-mediated multidrug resistance in cancer: A review of the past decade. Drug Resist Updat. 2015 January; 18C:1-17.doi:10.1016/j.drup; Shukla S, Ohnuma S, Ambudkar S V. Improving cancer chemotherapy with modulators of ABC drug transporters. Curr Drug Targets. 2011, 12(5), 621-30). The substrate of ABCG2 includes organic anion conjugate, nucleoside analogue, organic dye, tyrosine kinase inhibitor, anthracyclines, camptothecin-derived topoisomerase I inhibitor, methotrexate, flavonoid antineoplastic agents, etc. (Shukla S, Ohnuma S, Ambudkar S V. Improving cancer chemotherapy with modulators of ABC drug transporters. Curr Drug Targets. 2011, 12(5), 621-30). By inhibiting the expression of ABCG2 during tumor therapy, the efficacy of doxorubicin and mitoxantrone in breast cancer and irinotecan (camptothecin) in advanced colon cancer can be enhanced.
The Problems Currently Facing
Looking for drugs that effectively reverse ABCG2-mediated multidrug resistance has been a hot area in drug research and development (Assaraf Y G. The role of multidrug resistance efflux transporters in antifolate resistance and folate homeostasis. Drug Ressist Update, 2006, 9(4):227). Despite years of research, potential multidrug resistance reversal agents have not yet been available, the reasons for that may be: first, most of the available multidrug resistance inhibitors are small molecular compounds, which are all based on the regulation of the transport function of ABCG2. These multidrug resistance reversal agents, which have good in vitro regulatory effects, produce strong toxicity and side effects after entering complex human environment due to the changes in the metabolic pathways of antitumor drugs (Kathawala R J, Gupta P, Ashby C R Jr, Chen Z S. The modulation of ABC transporter-mediated multidrug resistance in cancer: A review of the past decade. Drug Resist Updat. 2015 January; 18C:1-17. doi:10.1016/j.drup). Second, there are few specific inhibitors against ABCG2, and some small molecule compounds are both inhibitors of ABCG2 and p-gp. Third, in recent years, various kinase inhibitors have been used in researches of reversing ABCG2-mediated multidrug resistance, which have a good in vitro effect of reversing drug resistance, but no clinical trials have been conducted. However, the tyrosin kinase inhibitors themselves produce drug resistance during the treatment of tumors, making the multidrug resistance developed in tumor more complicated.
In the study of antibody reversal of tumor drug resistance targeting ABCG2, by direct binding of antibodies to ABCG2 protein expressed on the tumor cell membranes, the drug efflux may be specifically blocked, and the drug accumulation effect may be increased. Currently, there is no domestic or foreign detailed report about that, and it is a new target for antibody drugs. Therefore, the research and development of antibodies targeting ABCG2 should have better potential application value in tumor treatments, which reflects in a better interpretation of mechanism of ABCG2-mediated multidrug resistance of tumor as well as efficient and specific blocking of ABCG2-mediated tumor intrinsic resistance, by targeting CSC, inhibiting tumor growth.
Taking into account the above factors, the present disclosure develops a monoclonal antibody, which targets ABCG2, capable of reversing ABCG2-mediated tumor drug resistance; in addition, a research of application in terms of tumor treatment and characterization of the antibody is conducted to complete the present disclosure.